The invention relates to recovery methods and apparatuses and in particular to methods and apparatuses for recovery by electrophoresis methods and devices. Specifically, it relates to a method and apparatus for the recovery of nucleic acids and other substances.
Electrophoresis of large charged molecules, such as deoxyribonucleic acid (DNA) fragments, has been an established laboratory method for approximately 20 years. In its most common form, an electric field is imposed between opposite ends of a thin slab of gel. A sample, containing a mixture of various charged molecules, such as DNA fragments, is introduced into one end of the gel. The electric field causes the DNA fragments to migrate to the opposite end of the gel. The velocity at which each DNA fragment migrates is dependent upon its mobility, a characteristic determined by the DNA fragment's length, shape, and other characteristics. Fragments having similar mobility will migrate as a band at a given velocity. With time, the sample will be separated into distinct bands, each composed of like fragments.
By means of staining or radioactive tagging, the various DNA fragments can be located and identified. As described above, electrophoresis is a simple and sensitive analytical technique. The recovery of the DNA fragments from the gel following electrophoresis has historically been difficult. Some of the methods that have been utilized are described hereinafter.
The crush and soak method: the gel material containing the DNA fragment to be recovered is excised from the gel slab and is ground or crushed. Soaking the crushed gel for long periods of time in a salt solution, extracts the DNA fragment from the gel. The resulting DNA fragment is then ethanol precipitated from the salt solution. The recovery efficiency by this method is quite low.
The melting agarose/chemical extraction method: the gel material containing the DNA fragment to be recovered is excised from the gel slab and is melted at about 65 degrees centigrade. Phenol extraction is followed by organic chemical purification. The recovery efficiency by this method is relatively low and the purification procedures are long and labor intensive.
Blitz blotting method: in this method, the entire gel slab is placed in contact against a sheet of DEAE cellulose paper, which is known for its ability to bind DNA fragments. This sandwich is then immersed in a buffer solution and by electrophoretic means, all the bands are transferred from the gel to the DEAE paper. This method has a relatively high recovery efficiency, but the apparatus is costly and cumbersome.
DEAE insertion in gel method: in this method, slots are cut in the gel immediately "downstream" of each of the bands. DEAE paper is inserted into the slots and by means of electrophoresis, the bands are driven inbo the DEAE paper. This method is extremely technique dependent. Cutting the slot deforms the electric field which causes varying portions of the band to be driven around the slot.
Electro-elution/dialysis membrane method: in this method, the gel material containing the DNA fragment to be recovered is excised from the gel slab and is immersed in the buffer solution in the apparatus. An electric field elutes the DNA fragment into the buffer and onto a dialysis membrane where it is recovered by washing. A major drawback is that all molecules above a certain size are deposited on the dialysis membrane. The DNA fragment may irreversibly bind to the membrane.
Gel decomposition/glass beads method: the gel material containing the DNA fraction is decomposed by chemical means and the resulting mixture, in a buffer, is poured over glass beads which are removed and washed. The DNA is eluted from the glass beads with a salt solution. Fair recovery efficiency with large molecules. Buffer conditions are very critical.
Each of the described prior art methods has disadvantages in either efficiency, ease, purity, or cost. Consequently, there is a need for a method combining the following characteristics: high quantitative recovery efficiency of biologically functional charged molecules free of contaminants; a simple setup; a rapid and unattended operation; and a relatively low cost. The present invention accomplishes this.
The present invention provides new and improved apparatus, as well as an improved method, for the recovery of charged molecules from gels which has a high quantitative recovery without contamination and the mechanism is rapid and convenient to use. As developed the apparatus is durable, inexpensive to obtain and to operate. For purposes of this specification the description refers to nucleic acids. It is to be understood, however, that it is within the scope and intent of the invention to utilize it for the recovery of other substances, such as proteins.
The apparatus consists of a plurality of transfer chambers, preferably of an inert and non-conductive plastics which are suitably supported in a vessel for containing an aqueous buffer solution, a plurality of filter discs for support of a layer of DEAE cellulose resin (one disc for each transfer chamber), a plurality of negative electrodes (one in each of the upper portion of the transfer chambers), a positive electrode for placement in the buffer which will surround the plurality of transfer chambers, and a power supply. Other refinements of the apparatus structure will be described hereinafter.
In operation, the gel containing the DNA to be recovered is excised from the gel slab and sliced into conveniently sized pieces for placement in the transfer chambers. The filter disc for supporting the layer of DEAE cellulose resin is in the bottom of each of the transfer chambers. The negative electrodes are placed in contact with the buffer in the upper portion of each of the transfer chambers. The positive electrode is placed in contact with the buffer that surrounds the plurality of transfer chambers.
The buffer permeates the filter and bed of DEAE to form a continuous conductive path for the flow of an electrical current between the electrodes. An orifice at the bottom of each of the transfer chambers restricts the electical current flow to the center of the bed of DEAE to assure passage of the electrical current through the DEAE.
When the power supply is activated, an electrical field is established in each transfer chamber. Positively charged molecules are attracted to each of the negative electrodes and vice versa. The negatively charged DNA fragments within the gel, under influence of the electrical field, begin to migrate through the gel and toward the path to the positive electrode.
After exiting the gel, the DNA fragments continue to migrate through the buffer toward the orifice. On passing through the DEAE, the DNA fragments are brought into intimate contact with the DEAE resin and are bound to the surface of the DEAE resin, which has a very specific binding affinity for DNA.
To recover the DNA, each of the transfer chambers is removed and allowed to drain. Following washing, the DNA is eluted from the DEAE by established procedures using the appropriate salt solution.
It is, therefore, an object of this invention to provide an apparatus for recovery of charged molecules from gels which has a high quantitative recovery without contamination.
It is also an object of this invention to provide an apparatus that is an improved electrophoresis device for the recovery of nucleic acids.
It is another object of this invention to provide an apparatus that has a simple setup.
It is still another object of this invention to provide an apparatus that has a rapid and unattended operation.
It is yet another object of this invention to provide an apparatus that is relatively low in cost.
Further objects and advantages of the invention will become more apparent in light of the following description of the preferred embodiments.